Methods for reducing ischemic injury of the heart via the sequential administration of monophosphoryl lipid A and adenosine receptor agents

ABSTRACT

Materials and methods for reducing or preventing ischemic damage of the heart are disclosed. A preferred embodiment of the invention comprises methods of sequential administration of a plurality of cardioprotective agents to patients suffering from ischernic damage or at risk for the same.

This application is a provisional of No. 60,062,737 filed Oct. 23, 1997

Pursuant to 35 U.S.C. §202(c) it is acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made in part with funds from the National Institutes of Health,Grant Number HL48225.

FIELD OF THE INVENTION

The present invention relates to methods for administering compounds toprotect the heart from ischemic injury. More specifically, the inventionprovides a novel combination of agents which act synergistically topotentiate their individual cardioprotective effects and thereby renderthe myocardium more resistant to ischemia.

BACKGROUND OF THE INVENTION

Several publications are referenced in this application by numerals inparentheses in order to more fully describe the state of the art towhich this invention pertains. Full citations for these references arefound at the end of the specification. The disclosure of each of thesepublications is incorporated by reference herein.

Ischemic preconditioning functions as an endogenous protective mechanismwhich enhances the ability of myocardium to withstand injury fromprologed ischemia. Transient ischemic events, which are sufficientlybrief to avoid irreversibly damaging myocardium, initiate unidentifiedbiochemical events which limit infarct size and the occurrence ofreperfusion arrhythmias following prolonged myocardial ischemias (5).

A series of studies have shown that it is possible to induce ischemicpreconditioning with a variety of pharmacological agents. Adenosine isreleased in large amounts during myocardial ischemia and mediatespotentially important protective functions in the cardiovascular system(1,4,5,7,9,14,17,18,19,25). Adenosine can precondition the heart withreduction in the size of myocardial infarction (4,5,9,14,17,18).Intracoronary administration of adenosine during reperfusion followingprolonged no-flow ischemia can also limit infarct size in the intactheart (1, 19).

Previous studies have shown that adenosine A₁ and A₃ receptor agonistscan precondition the heart when administered before the onset ofischemia (4, 5, 9, 14, 17, 18). Other studies have shown that adenosineA_(2a) receptor antagonists also enhance the cardioprotective effect ofpreconditioning (23). These agents effectively 1) reduce infarct size;and 2) improve left ventricular function when given during reperfusion(1, 19) or during both low-flow ischemia and reperfusion in isolatedperfused heart (6, 21, 22).

Monophosphoryl lipid A (MLA), a relatively non-toxic derivative ofendotoxin, has also been found to provide cardioprotection in a varietyof animal models (43-45) when administered prior to an ischemic event.The mechanism of myocardial protection mediated by MLA has not yet beendefinitively elucidated. Other effective pharmacological preconditioningagents include K⁺ATP channel openers and phorbol esters.

Intensive research efforts are currently focused on the development ofagents and methods for treating and preventing cardiac diseases. Thepresent invention is directed to such methods.

SUMMARY OF THE INVENTION

Methods are disclosed which may be used to advantage for preventingischemic damage of the heart.

The methods of the invention entail the combined administration of afirst cardioprotective agent and a second cardioprotective agent. Theagents act synergistically to potentiate their individualcardioprotective effects. Agents suitable for use as a firstcardioprotective agent are adenosine receptor agents, MLA or analogs orderivatives of MLA.

According to a preferred embodiment, the invention entails theadministration of monophosphoryl lipid A (MLA), or a synthetic analog,or derivative thereof to a patient prior to a surgical treatment.Following MLA treatment, a second cardioprotective agent is administeredthat potentiates the cardioprotective effect of MLA. The secondcardioprotective agent is delivered to the patient either before, duringor after surgery. Alternatively, the second cardioprotective agent maybe administered continuously throughout all of these periods. Theprotocols described above may be used for the treatment of patients atrisk for ischemic damage from myocardial infarction, angina, or surgicalcomplications.

Compounds suitable as second cardioprotective agents are adenosine,agonists at the A₁ and A₃ adenosine receptors, antagonists at the A_(2a)adenosine receptor, and K_(ATP) channel openers.

In an alternative embodiment, an adenosine receptor agonist isadministered as the first cardioprotective agent and MLA or syntheticanalogs thereof is administered as the second cardioprotective agent.

Methods of administration of the cardioprotective agents of theinvention include direct perfusion of the organ during surgery andintravenous administration. Additionally, these agents may beadministered to patients in solid form, e.g., tablets, in amountseffective to prevent or reduce ischemic damage to the heart.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the protective effect of pretreatment with MLAprior to prolonged ischemia in cardiac myocytes.

FIG. 2 is a graph showing that pretreatment with MLA enhances thecardioprotective effect of preconditioning ischemia in cardiac myocytes.

FIG. 3A is a graph showing that pretreatment with MLA enhances thepreconditioning effect mediated by adenosine in cultured myocytes (□,vehicle; ∘, MLA 30 ng/ml; ▪, MLA, 300 ng/ml). Cardiac ventricularmyocytes were subjected to classical preconditioning as describedherein.

FIGS. 3B and 3C are graphs showing the results obtained when myocyteswere treated with CCPA (FIG. 3B) or IB-MECA (FIG. 3C) for five minutesfollowed by incubation in adenosine- or agonist-free media for 10minutes prior to 90 minutes of ischemia. Data, plotted as percentage ofcells killed and the amount of CK released, represented the mean andstandard error of four experiments. * indicates significant differencefrom vehicle-treated myocytes at the concentrations of adenosineagonists indicated (t test, P<0.001).

FIG. 4 is a graph showing that pretreatment with MLA enhances thepreconditioning effect mediated by phorbol 12-myristate 13-acetate (PMA)(□, vehicle; ▪, MLA 300 ng/ml).

FIG. 5 is a graph showing that pretreatment with MLA enhances thepreconditioning effect mediated by pinacidil in cardiac myocytes (□,vehicle; ∘, MLA 30 ng/ml; ▪, MLA, 300 ng/ml).

FIG. 6 is a graph showing that pretreatment with MLA enhances thecardioprotective effect of the A₁ adenosine receptor agonist,2-chloro-N⁶-cyclopentyl-adenosine (CCPA), during prolonged ischemia (□,vehicle; ▪, MLA 300 ng/ml).

FIG. 7 is a graph showing that pretreatment with MLA enhances thecardioprotective effect of the A₃ adenosine receptor agonist,N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA), duringprolonged ischemia (□, vehicle; ▪, MLA 300 ng/ml).

FIG. 8 is a graph showing that pretreatment with increasingconcentrations of MLA enhances the cardioprotective effect of pinacidilduring prolonged ischemia (□, vehicle; ▪, MLA 300 ng/ml).

DETAILED DESCRIPTION OF THE INVENTION

Preconditioning with brief ischemia before a sustained period ofischemia reduces infarct size in the perfused heart. Ventricularmyocytes cultured from chick embryos retain many of the properties ofthe intact heart, which make them a useful model for a variety ofexperimental paradigms (33-39), including preconditioning (16, 23, 32).This model system has been utilized to develop novel combinations ofpharmcological agents which act synergistically to treat and preventischemic damage of the heart.

Monophosphoryl lipid A (MLA), a derivative of the gram-negativebacterial cell wall component lipopolysaccharide A (i.e. endotoxin),induces a glibenclamide-sensitive cardioprotective effect in isolated,intact heart models of ischemia-reperfusion. The data reveal thattreatment with MLA has a prolonged protective effect (31). The presentinvention is based on the hypothesis that MLA or MLA analogs effectcardioprotective action by enhancing the sensitivity of the cardiacmyocytes to preconditioning stimuli and to cardioprotective agents.These cardioprotective agents include agonists at the A₁ and A₃adenosine receptors, antagonists at the A_(2a) adenosine receptor,K_(ATP) channel openers and phorbol esters. MLA may also enhance theprotective effects of these agents during prolonged, infarct-producingischemia. These two novel cardioprotective mechanisms of MLA wereinvestigated and the results are provided hereinbelow.

The present invention demonstrates that pretreatment of myocytes withMLA alone prior to preconditioning ischemia can enhance thecardioprotective effect of the preconditioning, thus further decreasingmyocyte killing in response to an ischemic event. Furthermore, asynergistic cardioprotective effect is achieved by treatment with MLA inconjunction with adenosine, agonists of the A₁ and A₃ adenosinereceptors, antagonists of the A_(2a) adenosine receptor or a K_(ATP)channel opening compound.

The compounds of the present invention tend to produce certain unwantedside effects when administered individually at standard dosage. Becausethese compounds have been discovered to act synergistically when used incombination, subthreshold concentrations may be employed in practicingthe methods of the present invention. These subthreshold concentrationsshould induce fewer side effects and accordingly constitute animprovement in currently available methods for reducing ischemic damageto the heart.

The following definitions are provided to facilitate understanding ofthe present invention.

Preconditioning Ischemia

A brief ischemia which does not cause any cardiac damage, but protectsthe heart against damage during a subsequent prolonged ischemia. Theeffect of preconditioning ischemia is mediated by adenosine, which isreleased during the ischemia. Preconditioning may be induced by briefexposure to anoxic conditions for example.

Adenosine Receptors

A₁, A₃ and A_(2a) receptors are present on the myocardium (cardiacmuscle cells). While activation of the A₁ and A₃ receptors iscardioprotective, activation of the A_(2a) receptors is deleterious andcauses damage to the cardiac muscle cells.

Stable Angina

Condition observed in certain cardiac patients having a chronic risk formyocardial ischemia because of the chronic potential for an imbalancebetween the supply of oxygenated blood and the demand for it. Typically,such imbalance occurs during certain stresses, such as exercise,emotional stress or stress associated with a surgical procedure.

Unstable Angina

Condition observed in cardiac patients having frequent imbalance betweenthe supply of and the demand for oxygenated blood.

Post-myocardial Infarction Angina

Condition observed in patients who have recurrent ischemia following aheart attack.

Preconditioning Stimuli

Any drug, agent or treatment which induces preconditioning, such asbrief hypoxia, adenosine, pinacidil, phorbol ester, A₁ or A₃ adenosinereceptor agonists or A_(2a) receptor antagonists.

Myocardial Responsiveness

The myocardium can be treated so as to enhance the effectiveness andprotective effects of preconditioning. This enhancement leads to areduction in ischemic damage.

The following methods facilitate the practice of the present invention.

Preparation of Cultured Ventricular Cells

Ventricular cells were cultured from chick embryos 14 days in ovo(Spafas Inc., Storrs, Conn.) as previously described (16, 23). Cellswere cultivated in a humidified 5% CO₂-95% air mixture at 37 C. Allexperiments were performed on day 3 in culture, at which time cellsexhibited rhythmic spontaneous contraction. The medium was changed to aHEPES-buffered medium containing (mM) 139 NaCl, 4.7 KCl, 0.5 MgCl₂, 0.9CaCl₂, 5 HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) and2% fetal bovine serum, pH 7.4, 37 C. before exposing the myocytes to thevarious conditions at 37 C. Control myocytes were maintained inHEPES-buffered medium under room air. Hypoxia and glucose deprivationwere used to induce and simulate ischemia in the cultured cells. Hypoxiawas produced by placing the cells in a hypoxic incubator (NuAire) whereO₂ was replaced by N₂ as previously described (16,23).

Determination of Cell Injury

The extent of hypoxia-induced injury to the ventricular cell wasquantitatively determined by the percentages of cells killed. Toquantitate the % of cells killed, cells were exposed to a trypsin-EDTAHanks' balanced salt solution for 10 minutes for detachment after thevarious treatment, followed by centrifugation (300×g for 10 minutes) andresuspension in culture media for counting in a hemocytometer. Only livecells sedimented and the cells counted represented those that survived(46). None of the sedimented cells subsequently counted included trypanblue. In cells not exposed to hypoxia, trypsin-EDTA treatment followedby re-exposure to Ca²⁺-containing culture media did not cause theappearance of trypan blue-stained cells or any significant increase inproteins or creatine kinase (CK) in the trypsin-EDTA media following the300×g, 10 minute sedimentation of the cells. There was no protein in theculture media following a second 300×g centrifugation of resuspendedcells previously treated with trypsin-EDTA. Thus, trypsin treatment,re-exposure to Ca²⁺-containing media or 300×g sedimentation did notcause any significant damage to the control, normoxia-exposed cells.

The percentage of cells killed was calculated as the number of cellsobtained from the control group (representing cells not subjected to anyhypoxia or drug treatment) minus the number of cells from the treatmentgroup divided by number of cells in control group multiplied by 100%.

Chemical Reagents

2-chloro-N⁶-cyclopentyl-adenosine (CCPA) and pinacidil were purchasedfrom Research Biochemicals International (Natick, Mass.). Phorbol12-myristate 13-acetate (PMA) was purchased from Sigma.8-(3-chlorostyryl)caffeine (CSC),N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA) and2-chloro-N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA)were synthesized as previously described (40-42).

The following examples are provided to illustrate certain embodiments ofthe invention. They are not intended to limit the invention in any way.

EXAMPLE I

A cultured chick ventricular myocyte model was developed to investigatethe roles of a variety of compounds known to influence preconditioning.In accordance with the present invention, it has been discovered thatcertain agents, when applied in combination, act synergistically toreduce ischemic damage to the heart.

Pretreatment of ventricular myocytes with MLA was assessed to determinewhether it enhances the cardioprotective effect of preconditioningischemia. Hypoxia (O₂ <1%) and glucose deprivation were employed toinduce and simulate ischemia in the myocyte model of preconditioning(16, 23, 32).

Cardiac ventricular myocytes were cultured from chick embryos 14 days inovo and myocyte injury was induced as described hereinbelow. MLA wasadded to the media at the indicated concentrations for a 4 hour period24 hours prior to 90 minutes of simulated ischemia. The extent ofmyocyte injury, determined at the end of the 90 minute simulatedischemia, was assessed by the percentage of cells killed. The datarepresent the mean of 4 experiments. At 30 and 300 ng/ml MLAsignificantly lowered the percentage of cell death in response toprolonged ischemia. (One way ANOVA analysis and t test, p<0.01).

Exposure of cultured cardiac myocytes to 90 minutes of prolonged hypoxiacauses cell death. FIG. 1 illustrates that prior treatment with MLAsignificantly reduces cell death relative to the untreated controls.These data are in agreement with the cardioprotective effect of MLAtreatment seen in intact rat, rabbit, and canine hearts (43-45).

FIG. 2 shows that the extent of cardioprotection induced by a 5 minuteperiod of preconditioning ischemia (induced by hypoxia and the absenceof glucose, also termed simulated ischemia) is enhanced by pretreatmentwith MLA. As in FIG. 1, cultured myocytes were pretreated with theindicated concentrations of MLA for 4 hours. Twenty four hours later thecells were subjected to preconditioning ischemia, consisting of 5minutes of simulated ischemia. After a 10 minute recovery period innormal air, the cells were subjected to a prolonged period (90 minutes)of simulated ischemia, and the percentage of cells killed was assessed.When myocytes are pretreated with MLA 24 hours prior to preconditioningischemia treatment, there is a dose-dependent reduction in thepercentage of cells killed during a prolonged ischemic event. Thus,pretreatment with MLA in combination with classic preconditioningischemia provides a synergistic cardioprotective effect.

EXAMPLE II

Another objective of this study was the elucidation of the mechanismunderlying the observed enhanced responsiveness of cardiac myocytes topreconditioning stimuli following pretreatment with MLA. Thecardioprotective effects of preconditioning ischemia are mediated byadenosine receptors and the K_(ATP) channel. The following experimentswere performed to assess the most efficacious combination of agents forthe prevention of ischemic damage to the heart.

As shown in FIG. 3A, prior treatment with MLA enhanced the ability ofadenosine to elicit the preconditioning response. In this experiment,myocytes were treated with either the vehicle (0.04% propylene glycol,0.01% ethyl alcohol) or MLA 24 hours prior to a 5 minute exposure to 10μM adenosine. Adenosine was removed by replacement of the culture mediumwith adenosine-free medium, and the cells were exposed to normal roomair O₂ for 10 minutes, followed by 90 minutes of simulated ischemiatreatment. The sequential treatment of myocytes with MLA followed byadenosine significantly decreased the percentage of myocytes killed byprolonged ischemia, as compared to pretreatment with the vehiclefollowed by exposure to adenosine. Similarly, FIGS. 3B and 3C show thatprior treatment of myocytes with MLA followed by adenosine agonist, CCPAor by A3 agonist, IB-MECA, as a classical preconditioning stimuli, alsosignificantly reduced the percentage of myocytes killed by prolongedischemia as compared to pre-treatment with the vehicle followed byexposure to CCPA or IB-MECA alone, respectively.

An enhanced adenosine-mediated classical preconditioning response inmyocytes pre-exposed to MLA suggests that MLA acts to increase theresponsiveness of myocytes to the preconditioning effect of adenosine.Since the adenosine receptor mediates the preconditioning response toadenosine, such results may further suggest that MLA activates orpre-activates the adenosine receptor/G-protein/PKC/K_(ATP) channelpathway. Consistent with this hypothesis, the ability of the phorbolester phorbol 12-myristate 13-acetate (PMA) to mediate a preconditioningresponse was also enhanced by pretreatment with MLA. See FIG. 4. Thisenhancement by 300 ng/ml MLA pretreatment was significant over a rangeof PMA concentrations from 0.01 μM to 1 μM. The preconditioning effectsof CCPA or IB-MECA were also enhanced by pretreatment of myocytes withMLA. This enhancement was not observed in myocytes pretreated withvehicle alone (data not shown).

MLA pretreatment also enhanced the preconditioning response mediated bypinacidil. See FIG. 5. The data are consistent with the notion that MLAcan pre-activate the adenosine receptor, G protein, PKC, the K_(ATP)channel, or all four molecules. On the other hand, as the K_(ATP)channel is the most distal effector of the four, MLA may pre-activate oractivate the K_(ATP) channel directly to render the cardiac myocyte moreresponsive to the preconditioning effect of the K_(ATP) channel openerpinacidil.

EXAMPLE III

The experiments described in the previous examples suggest that MLA actsto enhance the preconditioning effects of various pharmacological agentssuch as adenosine and pinacidil.

The synergistic effects obtained using a combination of MLA and otherpreconditioning stimuli prompted studies assessing the activity ofagonists and antagonists of adenosine receptors following pretreatmentwith MLA.

The experimental protocol utilized in these studies entailedpretreatment of myocytes with MLA for a 4 hour period 24 hours prior toexposure of the cardiac myocytes to 90 minutes of simulated ischemia.During this 90 minute simulated ischemia period, severalcardioprotective agents were administered individually to cells andtheir protective effects were assessed.

FIG. 6 illustrates that prior treatment of myocytes with MLA enhancedadenosine A₁ agonist mediated protection during the 90 minute exposureto ischemia. In MLA pretreated myocytes, the presence of CCPA during the90 minute ischemia resulted in a greater reduction in the number ofcells killed as compared to the number of cells killed followingpretreatment with vehicle alone. Again the synergistic effects mediatedby the two cardioprotective agents are illustrated by the data.

Similar results were obtained when cells were pretreated with MLA thentreated with IB-MECA, an A₃ adenosine receptor agonist during the 90minute ischemia as described above for FIG. 6. See FIG. 7. Again theresults showed a synergistic cardioprotective effect mediated by thecombination of the two agents.

FIG. 8 shows the results obtained by pretreatment with MLA followed 24hours later, by exposure to pinacidil, a K_(ATP) channel opener, duringthe 90 minute ischemia. In MLA-pretreated myocytes, pinacidil caused agreater reduction in the number of cells killed as compared to vehiclepretreatment.

Additional experiments were performed to assess the differences incardioprotection observed when MLA was administered as the secondcardioprotective agent and an adenosine agonist, KATP channel opener orprotein kinase C activator was administered as the firstcardioprotective agent. The experiments were performed as follows:Cardiac myocytes were exposed to the first cardioprotective agent for 5minutes and the agent was removed. Cells were then exposed to a periodof simulated ischemia in the presence or absence of 300 ng/ml of MLA.The percentage of cells killed by simulated ischemia alone was 27±2%(n=9) in the absence of any treatment with any cardioprotective agent.When MLA was present during the simulated ischemia, the percentage ofcells killed was reduced from 27±2% to 14±1.2%. When cells were exposedto 5 minutes of 10 μM pinacidil, a KATP channel opener followed byexposure to simulated ischemia (in the absence of MLA), the percentageof cells killed was 12.3±1. When the pinacidil-treated cells wereexposed to simulated ischemia in the presence of MLA, the percentage ofcells killed was reduced from 12.3±1% to 6.3±1.2% (n=3). When cells wereexposed to 5 minutes of 1 μM PMA, a PKC activator, followed by exposureto simulated ischemia (in the absence of MLA), the percentage of cellskilled was 14±1. When PMA-treated cells were then exposed to simulatedischemia in the presence of MLA, the percentage of cells killed wasreduced to 10.5±1 (n=3). When cells were exposed to 5 minutes of 10 μMadenosine, followed by exposure to simulated ischemia (in the absence ofMLA), the percentage of cells killed was 12±1.4%. When adenosine treatedcells were then exposed to simulated ischemia, in the presence of MLA,the percentage of cells killed was reduced to 7.4±1.2% These resultsindicate that the cardioprotective agents of the invention are effectivein reducing ischemic damage of the heart regardless of the order inwhich they are administered to cells.

The data presented in the Figures illustrate the synergistic effectsmediated by the combination of MLA, adenosine receptor agonists andK_(ATP) channel openers in mediating cardioprotection. Accordingly,other agents known to act on the adenosine receptors are contemplatedfor use in the present invention. Earlier studies have revealed thatantagonists at the A_(2a) receptor mediate cardioprotecctive effects.Thus, the combined use of MLA or analogs thereof followed by treatmentwith an A_(2a) antagonist should also prove to be efficacious inreducing ischemic injury of the heart. Candidate compounds of thefollowing formula that bind and inhibit adenosine A_(2a) receptoractivation are set forth below in Table I.

R1, R3=methyl, ethyl, propyl, allyl

R7=H, alkyl (C1-C8)

Rα=H

TABLE I Affinities of 8 Styrylxanthine Derivatives in RadioligandBinding Assays at Rat Brain A₁ and A₂-Receptors (47) A₁/A₂ cmpd R₁ + R₃R₁ X ratio 15b Me Me H  41 17b Me Me 2-MeO  18 19b Me Me 3-MeO  64 20bMe Me 3-CF₃  25 21b Me Me 3-NO₂  11 22b Me Me 3-NH₂  30 23 Me Me3-(AcNH)  240 24 Me Me 3-(HOOC(CH₂)₂CONH)  250 25 Me Me 3-(t-BOC-NH)  3026 Me Me 3-(t-BOC)₂N]  15 27b Me Me 3-F  190 28 Me Me 3-Cl  520 29b MeMe 4-MeO  44 32b Me Me 3,4-(MeO)₂  70 33a Me H 3,5-(MeO)₂  25 33b Me Me3,5-(MeO)₂  >200 34b Me Me 3,5-F₂  230 35 Me Me 3,5-(MeO)₂-4-OH  19 36Me Me 4-AcO-3,5-(MeO)₂  93 37 Me Me 4-(4-PhCH₂O)-3,5-(MeO)₂  30 38 Me Me4-(4-NH₂-BuO)-3,5 (MeO)₂  36 39 Me Me 4-[4-(tBOC-NH)BuO]-3,5-(MeO)₂  4240 Me Me 4-(4-NH₂-trans-CH₂CH═  28 CHCH₂O-3,5-(MeO)₂ 41 Me Me4-(4-AcNH-trans-CH₂CH═  >50 CHCH₂O-3,5-(MeO)₂ 42 Me Me4-(4-t-BOC-NH-trans-CH₂CH═  >40 CHCH₂O-3,5-(MeO)₂ 43b Me Me 2,3,4-(MeO)₃ 34 44b Me Me 3,4,5-(MeO)₃  70 [>5600] 45b Et Me 3,4,5-(MeO)₃  34 46allyl Me 3,4,5-(MeO)₃  13 [>6700] 51b Pr Me 3-Cl  14 52b Pr Me3,4-(MeO)₂  19 [190] 53b Pr Me 3,5-(MeO)₂  110

Additional compounds contemplated for use as A_(2a) antagonists include:

Y=m-Br or p-SO₃H (DMPX derivative)

(ZM241385)

(SCH58261)

As mentioned previously, agonists at the A₁ and A₃ adenosine receptorsare also contemplated for use in the methods of the present invention.Suitable agonists of the following formula are set forth below in TableII.

COMPOUND A₁ A₃ (Reference) R₁ R (nm) A_(2a) (nm) (nm) Compound 4q(Reference 27) Ethyl

16 3940 30 Compound 4d (Reference 27) Ethyl

384 >10,000 54 Compound 37 (Reference 8) Ethyl

49 574 9.0 Compound 11 (Reference 28) Methyl

2060 66,300 1340 N⁶-cyclohexyl NECA (Reference 29) Ethyl

0.43 170 16

4q=N⁶-((3-iodophenyl)carbamoyl)adenosine -5′uronamide

4d=N⁶-((2-trifluoromethyl)carbamoyl)adenosine-5′uronamide

compound 37=N⁶-(4-Nitrobenzyl)adenosine-5′-N-methyluronamide

compound11=6-(O-Phenylhydroxylamino)purine-9-beta-ribofuranoside-5′-N-methyluronamide

N6-cyclohexyl NECA=N6-cyclohexyl 5′-N-ethylcarboxamidoadenosine

COMPOUND R A₁ (nm) A₂ (nm) A₃ (nm) Compound 8* (See immediately 0.85 2104 above.)

*N⁶-[4-[[[4-[2-aminoethyl)amino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl]adenosine

Other effective A3 agonists suitable for use in practicing the methodsof the present invention are set forth below:

EXAMPLE IV

The synergistic cardioprotective effect of MLA used in combination withA₃/A₁ agonists and a K_(ATP) channel opener has been demonstratedherein. Combinations of these agents may be used therapeutically inpatients who suffer from ischemic damage due to stable angina, unstableangina or post-myocardial infarction angina.

Several administration modalities may be utilized to treat patients withthe agents of the invention. These modalities are influenced bybioavailability factors. For example, if the compound is metabolized inthe liver or excreted in the bile, some of the active compound absorbedfrom the gastrointestinal tract will be inactivated by the liver beforeit can reach the general circulation and for distribution to the site ofaction. It is not believed that adenosine A₁/A₃ receptor agonists,adenosine A_(2a), receptor antagonists or K_(ATP) will be subject tothis first pass loss. Additionally, because the agonists of theinvention are polar and water soluble, it is expected that they willhave a small volume of distribution, and thus be readily eliminated bythe kidney. Moreover, binding of these agents to plasma proteins maylimit their free concentrations in tissues and at their locus of actionsince it is only the unbound drug which equilibrates across membranereceptor sites.

Another factor affecting bioavailability is the distribution of theagonists to tissues. Given the relatively small size of these compoundsand their water solubility, it is anticipated that the compounds willhave a relatively fast second phase of drug distribution. Thisdistribution is determined by both the blood flow to the particulartissue of the organ such as the heart, as well as the rate at which thecompounds diffuse into the interstitial compartment from the generalcirculation through the highly permeable capillary endothelium.

MLA or synthetic analogs thereof are lipophilic and will be administeredintravenously in a 10% ethanol, 40% propylene glycol water solution.Alternatively MLA or its analogs can be given orally in capsules ortablets. MLA or its analogs would be administered in doses ranging from1-20 μg/kg body weight. Patients may be perfused with the adenosinereceptor A₁ or A₃ agonists, adenosine A_(2a) receptor antagonists orK_(ATP) channel openers of the invention by dissolving them in normalsaline solution or using emulsifying agents or cosolvents followed byintravenous administration every four to six hours. Effective dosesusually range from 100 to 300 nM. For example, considering a 15 litervolume of distribution for a 70 kg patient, a loading dose ranging from0.5 to 1.5 mg is preferably used. Depending on the half-life of theagonists in the body, several doses, e.g., 1.5-4.5 mg may beadministered per day.

Alternatively, a time-release or slow-release preparation may beutilized which allows for periodic or constant release of the agonists,antagonists or channel openers over a given time period. This methodwould allow for a single dose of these agents in a given day. Methodsfor preparing such capsules are well known to those of skill in the artof drug delivery.

While the combined use of MLA with specific A₃ and A₁ agonists orK_(ATP) channel openers has been exemplified herein, other compoundshave been identified which have high affinity for both the A₃ and A₁receptor, also other compounds are known to those skilled in the artthat function to open the K_(ATP) channel. Additionally, analogs orderivatives of MLA are known. Any of these compounds or agents thereforemay also be used in the practice of the instant invention.

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While certain preferred embodiments of the present invention have beendescribed and specifically exemplified above, it is not intended thatthe invention be limited to such embodiments. Various modifications maybe made to the invention without departing from the scope and spiritthereof as set forth in the following claims.

What is claimed is:
 1. A method for preventing or reducing ischemicheart damage, in a patient in need of such treatment, comprisingadministering to said patient a first cardioprotective agent followed byadminstration of a second cardioprotective agent, said first and secondcardioprotective agents acting synergistically to protect the heart fromischemic damage.
 2. A method as claimed in claim 1, wherein said firstcardioprotective agent is selected from the group consisting ofmonophosphoryl lipid A, a synthetic analog of monophosphoryl lipid A,adenosine A1 agonists or adenosine A3 agonists.
 3. A method as claimedin claim 1, wherein said second cardioprotective agent is selected fromthe group consisting of adenosine A1 receptor agonists, adenosine A3receptor agonists, N⁶-((3-iodophenyl)carbamoyl)adenosine-5′uronamide,N⁶-((2-trifluoromethyl)carbamoyl)adenosine-5′uronamide,N⁶-(4-nitrobenzyl)adenosine-5′-N-methyluronamide,6-(O-phenylhydroxylaminopurine-9-beta-ribofuranoside-5′-N-methyluronamide,N⁶-cyclohexyl-5′-N-ethylcarboxamido)adensine,N⁶-[4-[[[4-[2-aminoethyl)amino]carbonyl]methyl]-anilino]carbonyl]methyl]phenyl]adenosine,MRS 584, MRS 479, MRS 537, MRS 1340, adenosine A2a receptor antagonists,8 styrylxanthine derivative compounds listed in Table I consisting ofcompounds 15b, 17b, 19b, 20b, 21b, 22b, 23, 24, 25, 26, 27b, 28, 29b,32b, 33a, 33b, 34b, 35, 36, 37, 38, 39, 40, 41, 42, 43b, 44b, 45b, 46,51b, 52b, 53b, PKC activators and K_(ATP) channel openers.
 4. A methodas claimed in claim 1, wherein said first cardioprotective agent isadministered to said patient by an administration means selected fromthe group consisting of intravenous administration, direct cardiacperfusion, or oral administration.
 5. A method as claimed in claim 1,wherein said second cardioprotective agent is administered to saidpatient by an administration means selected from the group consisting ofintravenous administration, direct cardiac perfusion, or oraladministration.
 6. A method as claimed in claim 1, wherein said patientis in need of said treatment due to the presence of angina selected fromthe group consisting of unstable angina or post-myocardial infarctionangina.
 7. A method as claimed in claim 1, wherein said cardioprotectiveagents are admininistered to said patient prior to a surgical procedurewhich may cause cardiac ischemic damage.
 8. A method as claimed in claim1, wherein said cardioprotective agents are admininistered to saidpatient during a surgical procedure which may cause cardiac ischemicdamage.
 9. A method as claimed in claim 1, wherein said cardioprotectiveagents are admininistered to said patient following a surgical procedurewhich may cause cardiac ischemic damage.
 10. A method for enhancingmyocardial responsiveness to preconditioning stimuli in a patient inneed of such treatment comprising administering to said patient a firstcardioprotective agent followed by adminstration of a secondcardioprotective agent, said first and second cardioprotective agentsacting synergistically to enhance myocardial responsiveness.
 11. Amethod as claimed in claim 10, wherein said first cardioprotective agentis selected from the group consisting of monophosphoryl lipid A, asynthetic analog of monophosphoryl lipid A, adenosine A1 agonists oradenosine A3 agonists.
 12. A method as claimed in claim 10, wherein saidsecond cardioprotective agent is selected from the group consisting ofadenosine A1 receptor agonists, adenosine A3 receptor agonists,adenosine A2a receptor antagonists, 8 styrylxanthine derivativecompounds listed in Table I consisting of compounds 15b, 17b, 19b, 20b,21b, 22b, 23, 24, 25, 26, 27b, 28, 29b, 32b, 33a, 33b, 34b, 35, 36, 37,38, 39, 40, 41, 42, 43b, 44b, 45b, 46, 51b, 52b, 53b, PKC activators andK_(ATP) channel openers.
 13. A method as claimed in claim 10, whereinsaid first cardioprotective agent is administered to said patient by anadministration means selected from the group consisting of intravenousadministration, direct cardiac perfusion, or oral administration.
 14. Amethod as claimed in claim 10, wherein said second cardioprotectiveagent is administered to said patient by an administration meansselected from the group consisting of intravenous administration, directcardiac perfusion, or oral administration.
 15. A method as claimed inclaim 10, wherein said patient is in need of said treatment due to thepresence of angina selected from the group consisting of unstable anginaor post-myocardial infarction angina.
 16. A method as claimed in claim10, wherein said cardioprotective agents are admininistered to saidpatient prior to a surgical procedure which may cause cardiac ischemicdamage.
 17. A method for reducing or preventing ischemic heart damage ina patient in need of such treatment, comprising the administration offirst and second cardioprotective agents, said first cardioprotectiveagent being selected from the group consisting of A1 adenosine agonistsor A3 adenosine agonists, K_(ATP) channel openers, and PKC activatorsfollowed by the administration of a second cardioprotective agentselected from the group consisting of monophosphoryl lipid A orsynthetic analogs of monophosphoryl lipid A.
 18. A method as claimed inclaim 17, wherein said cardioprotective agents are admininistered tosaid patient prior to a surgical procedure which may cause cardiacischemic damage.
 19. A method as claimed in claim 17, wherein saidcardioprotective agents are admininistered to said patient during asurgical procedure which may cause cardiac ischemic damage.
 20. A methodas claimed in claim 17, wherein said cardioprotective agents areadmininistered to said patient following a surgical procedure which maycause cardiac ischemic damage.
 21. A method as claimed in claim 17,wherein said first cardioprotective agent is administered to saidpatient by an administration means selected from the group consisting ofintravenous administration, direct cardiac perfusion, or oraladministration.
 22. A method as claimed in claim 17, wherein said secondcardioprotective agent is administered to said patient by anadministration means selected from the group consisting of intravenousadministration, direct cardiac perfusion, or oral administration.
 23. Amethod as claimed in claim 17, wherein said patient is in need of saidtreatment due to the presence of angina selected from the groupconsisting of unstable angina or post-inyocardial infarction angina. 24.A method as claimed in claim 17, wherein said K_(ATP) channel opener isnicorandil.
 25. A method as claimed in claim 17, wherein said firstcardioprotective agent is selected from the group consisting ofN⁶-((3-iodophenyl)carbamoyl)adenosine-5′uronamide,N⁶-((2-trifluoromethyl)carbamoyl)adenosine-5′uronamide,N⁶-(4-nitrobenzyl)adenosine-5′-N-methyluronamide,6-(O-phenylhydroxylaminopurine-9-beta-ribofuranoside-5′-N-methyluronamide,N⁶-cyclohexyl-5′-N-ethylcarboxamido)adensine,N⁶-[4-[[[4-[2-aminoethyl)amino]carbonyl]methyl]-anilino]carbonyl]methyl]phenyl]adenosine,MRS 584, MRS 479, MRS 537, and MRS
 1340. 26. A method for enhancingmyocardial responsiveness to preconditioning stimuli in a patient inneed of such treatment comprising administering to said patient a firstcardioprotective agent followed by adminstration of a secondcardioprotective agent, said first cardioprotective agent being selectedfrom the group consisting of A1 adenosine agonists or A3 adenosineagonists, K_(ATP) channel openers, and PKC activators followed by theadministration of a second cardioprotective agent selected from thegroup consisting of A2a antagonists, monophosphoryl lipid A or syntheticanalogs of monophosphoryl lipid A, said first and secondcardioprotective agents acting synergistically to enhance myocardialresponsiveness.
 27. A method as claimed in claim 26, wherein said firstcardioprotective agent is administered to said patient by anadministration means selected from the group consisting of intravenousadministration, direct cardiac perfusion, or oral administration.
 28. Amethod as claimed in claim 26, wherein said second cardioprotectiveagent is administered to said patient by an administration meansselected from the group consisting of intravenous administration, directcardiac perfusion, or oral administration.
 29. A method as claimed inclaim 26, wherein said patient is in need of said treatment due to thepresence of angina selected from the group consisting of unstable anginaor post-myocardial infarction angina.
 30. A method as claimed in claim26, wherein said cardioprotective agents are admininistered to saidpatient prior to a surgical procedure which may cause cardiac ischemicdamage.